PROJECT SUMMARY/ABSTRACT Congenital heart disease (CHD) is the most common birth defect. Among various CHDs, single ventricle phenotypes resulting from altered ventricular morphogenesis have the poorest clinical prognoses and include Tricuspid Atresia (TA; OMIM# 605067), Double Inlet Left Ventricle (DILV), Pulmonary Atresia (OMIM# 265150), and Hypoplastic Left Heart Syndrome (OMIM# 241550, 614435). The single ventricle heart presents with a series circuit such that systemic venous return to the right ventricle and pulmonary arteries combined with the flow from the pulmonary venous return into the left ventricle and out to the body is incompatible with survival. Currently, there is a poor understanding of the molecular mechanisms and cellular etiology causative of the many forms of single ventricle CHD. The Basic helix-loop-helix factor Hand2 is expressed within the myocardium of the second heart field (SHF), epicardium, cardiac neural crest cells, and endocardium. Hand2 deletion results in E10.5 embryonic lethality due to the decreased growth in SHF-derived cardiac structures. Mef2c-Cre deletion of Hand2 from SHF progenitor cells, results in TA/ DILV phenotypes. We have recently determined that the observed single ventricle phenotypes are a direct consequence of Hand2 function in the endocardium. The function of Hand2 within the endocardium has not been investigated. Our findings lead to a novel hypothesis that signaling from the developing endocardium is critical in determining the position of the ventricular septum, which may potentially explain the etiology of some single ventricle CHDs. We outline a strategy to both characterize Hand2 endocardial function, to identify the direct upstream endocardial signaling network for Hand2, and the Hand2-downstream transcriptional targets within the developing endocardium. Defining these endocardial signaling pathways and developmental programs regulated by Hand2 will shed light on the cell etiology of single ventricle phenotypes and on the molecular programs controlling ventricular septation, thus expanding the understanding of ventricular wall trabeculation, and septal morphogenesis.